﻿A new species of the genus Achalinus (Squamata, Xenodermatidae) from southwest Hunan Province, China

﻿Abstract A new species of xenodermid snake, Achalinusnanshanensis H. Li, L.-Q. Zhu, Z.-Q. Zhang & X.-Y. Mo, sp. nov., is described based on three specimens collected from Nanshan National Park and Tongdao County of southwest Hunan Province. This new species is genetically distinct amongst its congeners with the mitochondrial COI uncorrected p-distance ranging from 4.4% (in A.yangdatongi) to 17.7% (in A.meiguensis). In addition, this new species can be distinguished from its congeners by a combination of the following morphological characters: (1) dorsal scales with 23 or 25 rows throughout and strongly keeled; (2) tail relatively longer so that TaL/ToL = 0.215–0.248; (3) length of suture between internasals significantly longer than that between prefrontals, LSBI/LSBP = 1.66–1.84; (4) single loreal scale present; (5) SPL 6 in number, with the fourth and fifth contacting eye; (6) IFL 6 in number, with the first three touching the first pair of chin shields; (7) TMP is 2-2-4/2-2(3)-4, with the anterior pair elongated and in contact with the eye; (8) ventrals 2 + 147–158; (9) subcaudals 64–77, unpaired; (10) dorsal body brownish black, with a bright yellow neck collar extending to the head and abdomen in the occipital region. The recognition of the new species increases the number of described Achalinus species to 28, of which 21 are found in China.

During recent herpetological surveys in southwest Hunan, China, three Achalinus snake specimens were collected in Tongdao County and Nanshan National Park.The specimens exhibit the morphological characteristics of the genus Achalinus, which include a small, elongated, cylindrical body; strongly keeled, lanceolate scales with a metallic luster; and the lack of preocular and postocular scales, featuring a single loreal scale and temporals that are in direct contact with the eyes (Peters, 1869;Zhao et al. 1998;Zhao, 2006), but they could not be assigned to any known species.Extensive morphological examinations and further molecular analyses revealed that these specimens represent a separately evolving lineage within the genus Achalinus and can be distinguished from recognized congeners.We herein describe this overlooked Achalinus population as a new species, based on an iterative taxonomic approach.

Sampling
Three odd-scaled snake specimens were collected in Hunan Province, China: two specimens (HNNU230902, HNNU230903) were collected in Nanshan National Park, and one specimen (HNNU230901) was collected in Tongdao County (Fig. 1).The three specimens were collected in the field, fixed in 75% ethanol, and deposited in the Vertebrate Zoology Laboratory, College of Life Science, Hunan Normal University.For molecular analyses, 33 sequences were used, among which 30 (sequences 4-33 in Table 1) were obtained from GenBank which include 27 sequences of 23 Achalinus species.Additionally, sequences of Fimbrios klossi, Smith, 1921, Parafimbrios lao, Teynié, David, Lottier, Le, Vidal & Nguyen, 2015and Xenodermus javanicus, Reinhardt, 1836 were used as outgroups.Details are shown in Table 1.

Morphological examination
Morphological descriptions follow Zhao (2006).Abbreviations in this study are as follows: snout-vent length (SVL, length from tip of snout to anterior margin of the cloaca); tail length (TaL, length from posterior margin of cloaca to tip of tail); total length (ToL, length from snout tip to tail end; head length (HL, length from the tip of snout to the posterior margin of mandible; head width (HW, width from the widest part of the head in dorsal view); eye diameter (ED, diameter from the most anterior corner of the eye to the most posterior corner); loreal height (LorH, height from the highest part to the lowest part of the loreal in lateral view); loreal length (LorL, length from the most anterior loreal to the most posterior loreal in lateral view); length of the suture between internasals (LSBI); length of the suture between prefrontals (LSBP).Three characters were measured with a ruler to the nearest 1 mm: SVL, TaL, and ToL; other measurements were measured used digital calipers to the nearest 0.1 mm.The scale features and their abbreviations are as follows: loreals (Lor); supralabials (SPL); infralabials (IFL); number of chin shield pairs (Chins); infralabials touching the first pair of chin shields (IFL-1 st Chin); postoculars (PtO); temporals (TMP); supraoculars (SPO); temporals (TEM), number of anterior temporals that touch the eye (aTEM-Eye) (head bilateral scale counts are given as left/right), pre-ventral scales (PrV), ventral scales (VEN), subcaudal (SC), entire or divided state of the anal scales (Anal), dorsal scale rows (DSR) (counted at one-head-length behind the head, at midbody, and at one-headlength before the anal); the number of maxillary teeth (MT).We also make comparisons with other species of the genus Achalinus based on available literature (Peters 1869;Boulenger 1888Boulenger , 1908;;Van 1912;Pope 1935;Bourret 1937;Hu and Zhao 1966;Hu et al. 1975;Zong and Ma 1983;Ota and Toyama 1989;Zhao et al. 1998;Zhao 2006;Wang et al. 2019;Ziegler et al. 2019;Li et al. 2020;Luu et al. 2020;Miller et al. 2020;Hou et al. 2021;Huang et al. 2021;Ha et al. 2022;Yang et al. 2022Yang et al. , 2023;;Ma et al. 2023aMa et al. , 2023b;;Xu et al. 2023;Zhang et al. 2023).The sex was determined by the presence/absence of everted hemipenes.

Phylogenetic analyses
Genomic DNA was extracted from preserved liver tissue using the TIANamp Genomic DNA Kit.The fragment of the mitochondrial DNA gene encoding cytochrome c oxidase subunit I (COI) was amplified using the primer pairs Chfm4 and Chrm4 (Che et al. 2012).The polymerase chain reaction (PCR) was performed in 20 μL of reactant with the following cycling conditions: 95 °C for 4 min, 35 cycles of denaturing at 94 °C for 40 s, annealing at 53 °C for 40 s, and extending at 72 °C for 1 min, and a final extending step of 72 °C for 10 min.The PCR products were sequenced at Shanghai Map Biotech Co., Ltd.
The COI sequences (629 bp) were assembled using SeqMan in the DNASTAR software package (Burland 2000), and compared and aligned using MEGA 7 software (Kumar et al. 2018).The uncorrected pairwise distances (p-distance) were calculated in MEGA 7. MrBayes 3.2.4(Ronquist et al. 2012) was used to conduct the Bayesian inference analysis under the best-fitting model GTR + I + G4, which was selected by ModelFinder identified via AICc (Darriba et al. 2012).A maximum-likelihood analysis (Nguyen et al. 2015) was executed using IQ-TREE 2 under the best-fit model TIM3 + F + I + G4 selected by Modelfinder according to AICc.

Molecular analyses
The maximum-likelihood (ML) and Bayesian-inference (BI) analyses resulted in essentially identical topologies, which are integrated in Fig. 2 with the distances given in Table 2.The new species is nested within the genus Achalinus, and its affinity to A. yangdatongi, A. damingensis, A. ningshanensis, and A. hunanensis considerably strong supported (BI, PP = 0.85; ML, BS = 93%).In addition, the p-distance among all species within the genus ranges from 4.4-17.7%(Table 2), the minimum genetic distance between the new species and its congers is greater than the lowest one (3.2-3.4% between A. ningshanensis and A. hunanensis).Given that the Achalinus populations from Tongdao County and Nanshan National Park possess significant morphological differences from all known congeners, we describe it as a new species below.Etymology.The new species is named for on its type locality.We suggest the Chinese common name 南山脊蛇 (pin yin: Nán Shān Jǐ Shé) and the English common name Nanshan odd-scaled snake.
Diagnosis.The new species can be distinguished from other members of Achalinus by the following characteristics: (1) dorsal scales with 23 or 25 rows throughout and strongly keeled; (2) tail relatively longer so that TaL/ToL = 0.215-0.248;(3) length of suture between internasals significantly longer than that between prefrontals, LSBI/LSBP = 1.66-1.84;(4) single loreal scale present; (5) SPL 6 in number, with the fourth and fifth contacting eye; (6) IFL 6 in number, with the first three touching the first pair of chin shields; (7) TMP is  Description of holotype.Adult male with a total length of 461 mm (SVL 362 mm and TaL 99 mm), tail relatively long, TaL/ToL 0.215, body slender and cylindrical.Head distinct from neck, rostral small, triangular, only upper tip visible from above.Head length 10.95 mm, head width 7.25 mm.Length of suture between internasals much longer than that between prefrontals (LSBI 1.78 mm, LSBP 1.07 mm, LSBI/LSBP 1.66).Frontal pentagonal pointed backwards, much shorter than parietals; each parietal bordered with an elongated nuchal, with no preoculars and postoculars.Nostril at anterior part of nasal scale, posterior margin of nostril with a distinct nostril cleft.A single loreal scale present, extending from nasal scale to eye, distinctly wider than high.Eyes small, ED 1.11 mm.Two aTMP and four pTMP present; aTMPs elongated, upper one much smaller than the lower one; upper one in contact with eye, lower one also in contact with parietal scale.SPL 6 in number, the fourth and fifth in contact with the eye, the sixth longest.Two pairs of shields present, the first three in contact with first chin shield.One mental scale present, the first IFL in contact with each other after the mental scale, followed by another 5 IFL in contact with each other.Dorsal scales 23-23-23, strongly keeled; dorsum with no longitudinal vertebral stripe.VEN 155 in number.SC 64 in number, uniserial, anal entire.
Coloration of holotype in life.Scales possess a subtle iridescent quality.The dorsum's distinguishing characteristic is its reflective, brownish-black appearance, with a notable bright-yellow patch that extends to the head and abdomen in the occipital region.The first pair of chin shields displays black coloration at the front, while the second pair is entirely white.The eyes are uniformly black.The ventral side is prevalently grayish white, with the edges of the ventral scales gradually transitioning from grayish white to black.The ventral coloration of the tail mirrors that of the dorsum, featuring a brownish-black hue.
Coloration in preservative.(Figs 4,5) All scales retain a subtle iridescence.The coloration darkens as it transitions from the dorsum to the venter, with the dorsal surface of the body primarily appearing brownish black.Notably, the collar of the neck is a paler grayish white.
Distribution and habits.(Fig. 6) Achalinus nanshanensis sp.nov. is currently known from Hunan Province, China, and specifically from Nashan National Park, Shaoyang City, and Tongdao County in Huaihua City.It has a known elevational range of 300-1665 m above sea level.All three specimens were found during the night, with the holotype and one paratype found near a mountain   stream where the air temperature was 18 °C and the relative humidity was 80%.These individuals were close to shrubs in a subtropical broadleaved evergreen forest.They were found making their way from leaf litter to the stream.The other specimen was found in a bamboo forest near a steam.
The new species differs from A. pingbianensis Li, Yu, Wu, Liao, Tang, Liu & Guo, 2020 in having a separated loreal (vs loreal fused with prefrontal), more subcaudals (64-77 vs 56), LSBI significantly longer than LSBP (vs length of suture between internasals subequal to that between prefrontals), two anterior temporals in contact with the eye (vs only the upper anterior temporal in contact with the eye), fewer supralabials (6 vs 7), and a bright-yellow collar around the neck.
Notably, a molecular comparison between populations of A. ater in Guangxi, China, and at its type locality at Tam Dao in northern Vietnam.Similarly, limited research has been made comparing A. spinalis found in China and at the type locality.This situation prompts us to reconsider the distribution of A. ater and A. spinalis in China.
Recent research has continued to underscore the remarkably high diversity within the genus Achalinus, which has lead to the discovery of an increasing number of species.However, several factors contribute to the difficulty in accurately identifying snakes of this genus based solely on morphology.Achalinus species display a conservative morphology; sexual dimorphism has been identified (particularly larger TaL/TL in males, more VEN in females, and more SC in males) (Ziegler et al. 2019;Hou et al. 2021;Huang et al. 2021;Li et al. 2021;Zhang et al. 2023); additionally, the possible existence of juvenile dimorphism has been suggested (Zhang et al. 2023).Furthermore, due to their secretive life history and morphological similarities, many cryptic species may well be hidden in plain sight within known widely distributed species (Ziegler et al. 2019;Li et al. 2020;Luu et al. 2020;Miller et al. 2020;Hou et al. 2021;Yang et al. 2022;Ma et al. 2023a;Yang et al. 2023).This poses a considerable challenge to future efforts aimed at comparing and identifying new species.
Molecular methods have played a pivotal role in the rapid discovery of Achalinus species (Yang et al. 2023).In addition, there are A. yunkaiensis and A. sheni distributed in the same region of A. nanshanensis sp.nov., which indicates that further study is necessary to conduct by using different geographic populations and molecular methods to revise their evolutionary history.

Figure 1 .
Figure 1.Distribution of some species of the genus Achalinus, Red star: the type locality of A. nanshanensis sp.nov.(HNNU230902, HNNU230903): Nanshan Nation Park, Shaoyang City, Hunan Province, China; A. nanshanensis sp.nov.(HNNU230901): Tongdao County, Huaihua City, Hunan Province, China.Blue triangle: the type locality of A. yangdatongi in Xichou County, Yunnan Province; Black circle: the type locality of A. damingensis in Shanglin County, Guangxi Province.Purple diamond: the type locality of A. hunanensis in Hecheng District and Ningxiang County, Hunan Province.

Figure 2 .
Figure 2. Phylogenetic tree of the genus Achalinus inferred from CO1 gene fragments (629 bp) by maximum-likelihood analysis.The numbers above the branches represent the supporting values: SH-like approximate likelihood ratio test and Bayesian posterior probabilities (the ones lower than 50 are displayed as "-").Achalinus nanshanensis sp.nov. is highlighted in blue and A. yangdatongi in yellow.

Table 1 .
Localities, voucher information, COI GenBank accession numbers, and references for all samples used in this study.